High calcium foodstuffs for weight loss

ABSTRACT

The subject invention provides methods of inducing the loss of adipose tissue by providing a diet high in calcium. In one aspect of the invention, the calcium is provided in the form of dairy products. In yet another aspect of the invention, calcium is provided in the form of a dietary supplement, such as calcium carbonate, of vitamin supplements. Methods of suppressing [Ca 2+ ] i  levels in individuals are also provided. The subject invention also provides methods of stimulating lipolysis, inhibiting lipogenesis, and increasing the expression of white adipose tissue uncoupling protein 2 (UCP2). The subject invention also provides methods of increasing the core temperature of an individual.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a divisional application of U.S. patentapplication Ser. No. 10/066,057, filed Jan. 31, 2002, which is adivisional of U.S. patent application Ser. No. 09/654,357, filed Sep. 1,2000, which issued as U.S. Pat. No. 6,384,087, which are herebyincorporated by reference in their entirety, including all figures andtables.

BACKGROUND OF THE INVENTION

[0002] The regulation of body weight, and particularly body fat, inanimals is a complex process having enormous implications for the healthand well being of humans and other animals. Excess body weight and/or anexcess of body fat relative to lean body mass has been associated with awide range of health problems including coronary artery diseases,stroke, and diabetes.

[0003] It has been estimated that half of all Americans are overweight.Within the United States about 24% of men and 27% of women defined asmildly to severely obese. Individuals 20% over ideal weight guidelinesare considered obese. Obesity is classified as mild (20-40% overweight),moderate (41-100% overweight), and severe (>100%) overweight. Severeobesity is relatively rare, affecting less than 0.5% of all obeseindividuals and about 0.1% of the total population.

[0004] Obesity is not just a problem for humans. Many animals alsosuffer adverse consequences related to obesity. For example,approximately 10 to 40% of cats receiving veterinary care have beenreported to be overweight. Factors contributing to feline obesityinclude a sedentary lifestyle, confinement to indoors, and neutering.Obese cats have a greater risk for certain diseases includingosteoarthritis, ligament injuries, perineal dermatitis, diabetesmellitus, cardiomyopathy, and urologic syndrome. Therefore, it iscritical to maintain a healthy weight in order to minimize disease risk.(See, U.S. Pat. No. 6,071,544.)

[0005] Obesity in humans is treated by a variety of means ranging fromsurgical procedures (gastric bypass) for the severely obese to diettherapy, behavior modification, and medication for the mildly tomoderately obese. Management of moderate and mild obesity is typicallyperformed by the individual and commercial organizations which providebehavior modification programs and, in some cases, prepackaged diets.The medical community recommends that diet treatments be administeredunder medical supervision.

[0006] The range of treatments for obesity reflects the complexity ofthe processes involved in weight regulation and the current lack ofunderstanding of these processes. Recent reports have even implicatedviruses as a possible causative factor in obesity (U.S. News and WorldReport, Aug. 7, 2000). There are also numerous reports of possiblegenetic bases for a predisposition to obesity. Moll et al. have reportedthat, in many populations, obesity seems to be controlled by a fewgenetic loci (Moll, et al., Am. J. Hum. Gen. 49:1243 (1991)). Inaddition, human twin studies strongly suggest a substantial geneticbasis in the control of body weight, with estimates of heritability of80-90% (Simopoulos, A. P. and Childs, B., eds., 1989, in “GeneticVariation and Nutrition in Obesity,” World Review of Nutrition andDiabetes, 63, S. Karger, Basel, Switzerland; Borjeson, M., Acta.Paediatr. Scand. (1976) 65:279-287).

[0007] Recombinant agouti protein, an obesity gene product, stimulatesCa²⁺ influx in a variety of cells. Agouti also stimulates the expressionand activity of fatty acid synthase (FAS), a key enzyme in de novolipogenesis, and inhibits basal and agonist-stimulated lipolysis inhuman and murine adipocytes via a Ca²⁺-dependent mechanism. Theseeffects can be mimicked in the absence of agouti by either receptor orvoltage-mediated Ca²⁺ channel activation and inhibited by a Ca²⁺ channelantagonist, such as nifedipine.

[0008] Recent data demonstrated that 1, 25-dihydroxyvitamin D(1,25-(OH)₂-D) causes a significant and sustained increase inintracellular calcium concentrations ([Ca²⁺]_(i)) in primary culturedhuman adipocytes and a corresponding inhibition of lipolysis (Zemel, etal., FASEB J. (2000) 14:1132-1138). Increasing dietary calciumsuppresses [Ca²⁺]_(i), inhibits 1,25-(OH)₂-D, and subsequentlysuppresses adiposity by stimulation of lipolysis and inhibition oflipogenesis. Consistent with these findings, increasing dietary calciumfrom 400 to 1000 mg/day resulted in a 4.9 kg reduction of body fat inhumans over the course of one year (Zemel, et al., FASEB J. (2000)14:1132-1138). Dietary calcium can also attenuate the diet-induceddevelopment of adiposity and promote weight loss in established obesity(Shi, H., et al., FASEB J. (2000) 555.3 (abstract)).

BRIEF SUMMARY OF THE INVENTION

[0009] The subject invention provides materials and methods for treatingor avoiding obesity in humans and other animals. Advantageously, thematerials and methods of the subject invention can be used to easily andefficiently achieve weight loss and/or prevent weight gain. In apreferred embodiment, the obesity-control benefits of the subjectinvention are achieved by providing a diet high in calcium. In oneaspect of the invention, individuals are maintained on a restrictedcaloric diet.

[0010] In a specific embodiment of the subject invention, calcium isprovided in the form of dairy products. In yet another aspect of theinvention, calcium is provided in the form of a dietary supplement, suchas calcium carbonate, or vitamin supplements.

[0011] The subject invention also provides methods of stimulatinglipolysis, inhibiting lipogenesis, and increasing the expression ofwhite adipose tissue uncoupling protein 2 (UCP2). The subject inventionalso provides methods of increasing the core temperature of anindividual as well as methods of diagnosing, treating, and/or monitoringobesity. Methods of suppressing [Ca²⁺]_(i) levels in individuals arealso provided.

[0012] The instant invention also provides methods of attenuating weightgain and adiposity in children and controlling weight gain in childrenby increasing the amounts of dietary calcium consumed by the children.

BRIEF DESCRIPTION OF THE DRAWINGS

[0013] FIGS. 1A-B show the effects of 1,25-(OH)₂-D (left panel) andparathyroid hormone (right panel) on intracellular calcium in humanadipocytes.

[0014]FIG. 2 depicts the effects of 1,25(OH)₂-D on forskolin-stimulatedlipolysis in human adipocytes. Forskolin treatment resulted in a˜two-fold increase in glycerol release; data shown are normalized to100% for the forskolin-treated group. (n=6/group; P<0.001).

[0015]FIG. 3 illustrates the effects of calcium and dairy products on 6wk weight gain in transgenic mice expressing agouti in adipose tissueunder the control of the aP2 promoter.

[0016]FIG. 4 depicts the effects of calcium and dairy products on coretemperature in transgenic mice expressing agouti in adipose tissue underthe control of the aP2 promoter.

[0017] FIGS. 5A-C illustrate the effects of calcium and dairy productson adipocyte fatty acid synthase activity (upper panel), fatty acidsynthase mRNA (middle panel) data are expressed as fatty acid synthase(FAS: actin ratio) and lipolysis in transgenic mice expressing agouti inadipose tissue under the control of the aP2 promoter. Data are expressedas mean±standard deviation (n=10/group). Statistical significance is asindicated in each panel.

[0018] FIGS. 6A-B depict the effects of calcium and dairy products onfasting plasma glucose and insulin levels in transgenic mice expressingagouti in adipose tissue under the control of the aP2 promoter. The toppanel depicts insulin and the bottom panel depicts glucose.

[0019] FIGS. 7A-C show the effects of 6-week consumption of basal lowcalcium (0.4%), high fat, high sucrose diet on body weight (top panel),total fat pad mass (middle panel) and basal adipocyte [Ca²⁺]_(i) (lowerpanel) in aP2-agouti transgenic mice. Data are expressed as mean±SE(n=8). *p <0.001 vs. before administration.

[0020] FIGS. 8A-C show the effects of 6-week administration of highcalcium diets on weight reduction (FIG. 8A, upper panel), fat pad mass(FIG. 8B, middle panel) and basal adipocyte [Ca²⁺]_(i) (FIG. 8C, lowerpanel) in energy-restricted (70% of ad lib) aP2-agouti transgenic mice.Data are expressed as mean±SE (n=8).

[0021] FIGS. 9A-C show the effects of 6-week administration of highcalcium diets on fatty acid synthase activity (FIG. 9A, upper panel),fatty acid synthase mRNA (FIG. 9B, middle panel) and lipolysis (FIG. 9C,lower panel) in energy-restricted (70% of ad lib) a Pe-agouti transgenicmice. Glycerol release is used to measure lipolysis. Data are expressedas mean±SE (n=8).

[0022] FIGS. 10A-B show the effects of 6-week administration of highcalcium diets on abdominal adipose tissue uncoupling protein 2 (UCP2)expression in energy-restricted (70% of ad lib) a P2-agouti transgenicmice. Upper panel (FIG. 10A): a representative northern blot from 8replicates; lower panel (FIG. 10B): quantitative analysis of UCP2/actinmRNA density units. Data are expressed as mean±SE (n=8). * p<0.05 vs.basal diet.

[0023]FIG. 11 shows the effects of dietary calcium on plasma insulinlevels. Data are expressed as mean±SE (n=8). * p<0.001 vs. before basaldiet treatment; +p<0.001 vs. ad libitum.

DETAILED DISCLOSURE OF THE INVENTION

[0024] The subject invention provides materials and methods for treatingor avoiding obesity in humans and other animals. Advantageously, thematerials and methods of the subject invention can be used to easily andefficiently achieve weight loss and/or prevent weight gain. In apreferred embodiment, the body weight benefits of the subject inventionare achieved by providing a diet high in calcium.

[0025] In a preferred embodiment, the subject invention provides methodsof inducing the loss of adipose tissue in a human or other animal byincreasing the ingestion of calcium. In one aspect of the invention, thecalcium is provided in the form of a dairy product, such as milk,yogurt, or cheese. In another aspect of the invention, calcium isprovided in the form of a dietary supplement, such as calcium carbonateor vitamin supplements. In yet another aspect of the invention,individuals are maintained on a restricted caloric diet.

[0026] Also provided are methods of decreasing intracellular calciumconcentrations ([Ca²⁺]₁), stimulating lipolysis , inhibitinglipogenesis, and increasing the expression of white adipose tissueuncoupling protein 2 (UCP2). The subject invention also provides methodsof increasing the core temperature of an individual (thermogenesis).Each of these methods is practiced by increasing the amount of calciumingested by an individual and, optionally, restricting the caloricintake of the individual.

[0027] Also provided are novel and advantageous methods of restoringnormal body fat ratios in women post partum. In one such embodiment, themethods are practiced by identifying an individual who has recentlygiven birth to a child and increasing the amount of dietary calciumconsumed by the individual.

[0028] Methods of preventing or reducing the regain of weight lost afteran initial period of dieting are also provided by the instant invention.This method can be practiced by identifying an individual who has lostweight as a result of a previous diet and increasing the amount ofdietary calcium consumed by the individual.

[0029] Also provided are methods of decreasing the levels ofcalcitrophic hormones (1,25-(OH)₂-D) in an individual by increasing theamount of dietary calcium consumed by the individual.

[0030] The subject invention also provides methods of reducing the riskof obesity in a child by increasing the amount of calcium consumed bythe child. Calcium intake can be accomplished by, for example,increasing the intake by the child of dairy products or other productscontaining high levels of calcium.

[0031] Also provided are methods of reducing, attenuating, or treatingobesity comprising decreasing the levels of calcitrophic hormones(1,25-(OH)₂-D) in an individual by administration of a therapeuticallyeffective amount of a 1,25-(OH)₂-D antagonist, for example dietarycalcium. Other 1,25-(OH)₂-D antagonists include 1,25-(OH)₂-Dneutralizing antibodies; soluble 1,25-(OH)₂-D receptor; fusion proteinscomprising the 1,25-(OH)₂-D receptor (for example soluble forms of the1,25-(OH)₂-D receptor fused to Ig heavy chains or moieties whichpreferentially target adipocytes); chemical compounds; and compoundscontaining calcium, such as calcium carbonate.

[0032] The subject invention also provides methods of reducing,treating, or attenuating obesity in an individual comprising theadministration of therapeutically effective amounts of 1,25-(OH)₂-Dreceptor antagonists. Examples of such antagonists include antibodieswhich block the ligand binding site of the receptor; chemical compoundantagonists of the receptor; and analogs, homologs, or isomers of1,25-(OH)₂-D which specifically bind to the 1,25-(OH)₂-D receptor butwhich antagonize the function of the receptor, for example1-β,25-dihydroxyvitamin D.

[0033] Calcium provided to an individual in accordance with the subjectinvention may take the form of, for example, a dairy product (forexample milk, non-fat dry milk, yogurt, cheese, cottage cheese, icecream or frozen yogurt), a nutrient supplement (such as calciumfortified vitamins and liquids supplemented with calcium), foodstuffssupplemented with calcium, or other foods high in calcium (for example,salmon, beans, tofu, spinach, turnip greens, kale, broccoli, waffles,pancakes, or pizza).

[0034] In a preferred embodiment, a therapeutically effective amount ofdietary calcium is provided to an individual. A therapeuticallyeffective amount of dietary calcium is an amount of calcium sufficientto induce weight loss, prevent weight gain, and/or increase themetabolic consumption of adipose tissue in a mammal.

[0035] Fusion proteins comprising the 1,25-(OH)₂-D receptor may be madeaccording to methods known in the art. An exemplary type of fusionprotein comprises the soluble form of the 1,25-(OH)₂-D receptor fused toIg heavy chains according to the teachings of Capon et al. (U.S. Pat.Nos. 5,565,3375 and 5,336,603, hereby incorporated by reference in theirentireties). In other embodiments, at least one 1,25-(OH)₂-D receptor(Nemere, et al., J. Bone Miner. Res. (1998) 13:1353-59; Fleet, et al.,Nutr. Rev. (1999) 57:60-64) is incorporated into liposomes which arepreferentially targeted to adipocytes using, for example, acylationstimulating protein (ASP) (Kalant et al., Clin. Invest. Med. (1995) 18(Supp. B:B10); Maslowska, et al., Int. J Obesity (1998) 22:S108;Maslowska, et al., Acylation Stimulating Protein (ASP): Role in AdiposeTissue, in Progress in Obesity Research: 8, (1999), Ed. B. Gut-Grand andG. Ailhaud, John Libbey & Co.). In other embodiments, soluble forms ofthe 1,25-(OH)₂-D receptor may be coupled to adipocyte targeting agentssuch as the ASP. Soluble forms of the 1,25-(OH)₂-D receptor may beproduced from the membrane bound form of the Vitamin D receptoraccording to methods known in the art. Coupling of one or more soluble1,25-(OH)₂-D receptors to one or more adipocyte targeting agents may beaccomplished recombinantly or using chemical crosslinking compounds,such as those sold by Pierce (Rockford, Ill.).

[0036] The term “individual” includes animals of avian, mammalian, orreptilian origin. Mammalian species which benefit from the disclosedmethods include, and are not limited to, apes, chimpanzees, orangutans,humans, monkeys; domesticated animals (pets) such as dogs, cats, guineapigs, hamsters, Vietnamese pot-bellied pigs, rabbits, and ferrets;domesticated farm animals such as cows, buffalo, bison, horses, donkey,swine, sheep, and goats; exotic animals typically found in zoos, such asbear, lions, tigers, panthers, elephants, hippopotamus, rhinoceros,giraffes, antelopes, sloth, gazelles, zebras, wildebeests, prairie dogs,koala bears, kangaroo, opossums, raccoons, pandas, giant pandas, hyena,seals, sea lions, and elephant seals. Reptiles include, and are notlimited to, alligators, crocodiles, turtles, tortoises, snakes, iguanas,and/or other lizards. Avian species include, and are not limited to,chickens, turkeys, pigeons, quail, parrots, macaws, dove, Guinea hens,lovebirds, parakeets, flamingos, eagles, hawks, falcons, condor,ostriches, peacocks, ducks, and swans.

[0037] Each of the methods discussed above may further compriserestricting the caloric intake of an individual. Additionally, dietaryproducts containing high levels of calcium may be provided to theindividual in conjunction with a dietary plan. The dietary products maybe provided to the individual on a regular or scheduled basis or ondemand by the individual.

[0038] The methods of the subject invention may further compriseanalysis of an individual's dietary intake. This analysis may beaccomplished by analysis of the foods consumed by the individual asrecorded on questionnaires regarding dietary intake or dietary logscompleted by the individual. The logs or questionnaires may be inelectronic or paper form.

[0039] Dietary intake information may be obtained by asking anindividual questions in person or over the internet. In this aspect ofthe invention, questions can be posed to the individual regardingdietary habits and/or food consumption. The questions may be transmittedover the internet. Alternatively, forms or dietary logs can betransmitted to the customer over the internet. In either aspect of theinvention, the information recorded by the individual can be returned tothe web site by way of the internet. Dietary intake may be analyzed by acomputer receiving the information from the individual through theinternet.

[0040] The analysis of the individual's dietary intake may be performedby a computer after input of the data related to food consumption by theindividual. The foods consumed by the individual, as well as theamounts, are compared to a database containing the nutritive values ofthe foods and the nutritional composition of the diet of the individualis provided. After analysis of the nutritional composition of the foodsingested by the individual, the amount of calcium consumed by theindividual is provided. Recommendations regarding increases in theamount of calcium consumed by the individual, as well as sources ofdietary calcium, may be provided from a database which compares theamount of calcium consumed with that found to optimize or induce weightloss.

[0041] In some instances, the caloric intake of an individual may beunmodified and caloric intake may be ad lib. In other instances, it maybe desirable to reduce the caloric intake of the individual as part ofthe dietary plan. In one embodiment, the range of caloric intake of theindividual is based upon gender. Caloric intake can range from about 200to about 2500 kcal per day. In a preferred embodiment, the range ofcaloric intake is about 300-2400 kcal per day, preferably 500-2200 kcalper day, more preferably 700-2000 kcal per day, even more preferably900-1700 kcal per day, and most preferably 1100-1500 kcal per day.

[0042] The weight/height ratio may be calculated by obtaining the weightof an individual in kilograms (kg) and dividing this value by the heightof the individual in meters. Alternatively, the weight/height ratio ofan individual may be obtained by multiplying the weight of theindividual in pounds (lbs) by 703 and dividing this value by the squareof the height of the individual (in inches (in)). These ratios aretypically referred to as BMI. Thus, BMI=kg/m² or BMI=(lbs.×703)/(in)².

[0043] Where BMI is utilized as a measure of obesity, an individual isconsidered overweight when BMI values range between 25.0 and 29.9.Obesity is defined as BMI values greater than or equal to 30.0. TheWorld Health Organization assigns BMI values as follows: 25.0-29.9,Grade I obesity (moderately overweight); 30-39.9, Grade II obesity(severely overweight); and 40.0 or greater, Grade Imi obesity(massive/morbid obesity). Using weight tables, obesity is classified asmild (20-40% overweight), moderate (41-100% overweight), and severe(>100%) overweight. Individuals 20% over ideal weight guidelines areconsidered obese. Individuals 1- 19.9% over ideal weight are classifiedas overweight.

[0044] Information related to the benefits of maintaining a normalweight, or optionally a normal weight/height ratio, dietary plan(s)containing high levels of calcium and printed matter disclosing thebenefits of a high calcium diet may, optionally be provided inelectronic or printed form and may be stored in a database.

[0045] A further aspect of the subject invention provides methods forpromoting good health by providing a product with calcium wherein theprovision of the product is accompanied by information regarding thebenefits of the consumption of calcium with respect to the control ofobesity. The calcium-containing product which is provided may be adietary product such as, for example, cereals, milk and other dairyproducts, vegetables and other foodstuffs which are either naturallyhigh in calcium or which are fortified with calcium. The product withcalcium may also be a dietary supplement in the form of, for example, apill or liquid.

[0046] The information regarding the benefits of the consumption ofcalcium with respect to the control of obesity would typically include,for example, a written or verbal explanation that the consumption ofcalcium is associated with weight loss and/or the prevention of weightgain. This information can be presented in such a fashion so that apotential purchaser and/or user of a product which contains calciumwould understand that the consumption of the product with calcium couldcause weight loss or reduce weight gain, and that the weight loss orreduction in weight gain from the use of the product would be directlyattributable, at least in part, to the calcium present in the product.

[0047] In accordance with the subject invention, the informationregarding the obesity-control benefits of a calcium-containing productmay directly accompany the product. Thus, the information may be, forexample, printed on a cereal box, a milk container, a cheese package, oran ice cream carton. The information may also be distributed throughbroader channels of communication including, for example, newspapers,magazines, direct mailings, radio, television, the internet, andbillboards. Other distribution channels include verbal communication,pamphlet distribution, print media, audio tapes, magnetic media, digitalmedia, audiovisual media, advertising, electronic mail, braille,electronic media, banner ads (including Internet banner ads and aircrafttowed banners), fiber optics, and laser light shows. In a preferredembodiment, the information thus distributed regarding the benefits ofcalcium with regard to weight loss would identify a particular productwith calcium which could be ingested in order to receive the obesitycontrol benefits of calcium. The particular product could be identifiedby, for example, a trade name.

[0048] In a preferred embodiment, the obesity-control benefits ofcalcium are communicated by the provider of a particularcalcium-containing product. The provider may be, for example, the ownerof the trade name for the product. The information regarding theobesity-control benefits of calcium may also describe the benefits of ageneral class of products which contain calcium. Thus, the informationmay describe, for example, the obesity-control benefits of diaryproducts which contain calcium. This information could be distributedby, for example, a trade association. Similarly, the information maydescribe the obesity-control benefits of dietary supplements whichcontain calcium.

[0049] The obesity-control information provided according to the subjectinvention may be provided with, or without, information relating toother health benefits which may be attributed to calcium or which may beattributed to other components of the product which contains calcium.Thus, the obesity-control information may be accompanied by informationconcerning the benefits of calcium with respect to osteoporosis.

[0050] In a specific embodiment, information regarding calcium'sobesity-control benefits would be practiced by a commercial entityhaving a financial interest in the sale of a product, or a class ofproducts, which contain calcium. This would not typically be, forexample, a doctor providing a specific patient with medical adviceregarding a diet high in calcium without reference to particular tradenames of products. This would also not typically include scientificarticles describing research evaluating the obesity-control benefits ofcalcium.

[0051] The subject invention also provides articles of manufactureuseful in stimulating the metabolic consumption of adipose tissue. Sucharticles of manufacture would typically comprise foodstuffs and printedmaterials disclosing the obesity-control advantages of high calciumdiets. In one aspect of the invention, the printed materials may be inthe form of pamphlets, package inserts, or as part of the package. Inanother aspect of the invention, the information may be embossed orimprinted on the foodstuff. Information so provided may include theamounts of calcium contained within the foodstuff, recommended levels ofcalcium intake necessary for the metabolically-assisted loss of adiposetissue, recommended BMI values, or recommended heights and weights forindividuals. The foodstuff may be a solid or liquid. In one aspect ofthe invention, liquids with increased levels of calcium are provided asa dietary supplement for the treatment of obesity or stimulation of themetabolic consumption of adipose tissue. In this aspect of theinvention, the liquids may be color coded to indicate the amounts ofcalcium contained therein. By way of example, food coloring additivesare introduced into the liquid to indicate the amounts of calciumcontained therein. A green colored liquid may contain 100 mg of calciumper serving, a yellow liquid may contain 200 mg of calcium per serving,and a magenta liquid may contain 500 mg of calcium per serving.

EXAMPLE 1 Regulation of Adiposity by Dietary Calcium Materials andMethods

[0052] Isolation and Culture of Human Adipocytes

[0053] Human subcutaneous adipose tissue was obtained from patients withno known history of metabolic disorders undergoing abdominal plasticsurgery. Adipocytes were isolated by washing, mincing, collagenasedigestion, and filtration as described previously and cultured inDulbecco's modified Eagle's medium supplemented with 1% fetal bovineserum, penicillin (100 IU/ml), streptomycin (100 μg/ml), and gentamicin(50 μg/ml). Cells were cultured in suspension and maintained in a thinlayer at the top of the culture media, which was changed every day.Cells were studied about 72 hours after isolation and were serum-starvedprior to study.

[0054] Animal and Diets

[0055] To evaluate the role of dietary calcium in regulating adiposityin vivo, transgenic mice expressing agouti specifically in adipocytesunder the control of the aP2 promoter were studied. These animalsexhibit a normal pattern of leptin expression and activity similar tothat found in humans and exhibit a human pattern (adipocyte-specific) ofagouti expression. These mice are useful models for diet-induced obesityin that they are not obese on a standard AIN-93G diet, but become obesein response to hyperinsulinemia induced by either insulin administrationor high sucrose diets. Male aP2-agouti transgenic animals from ourcolony were placed at 6 wk of age on a modified AIN 93-G diet withsuboptimal calcium (0.4%), sucrose as the sole carbohydrate source, andfat increased to 25% of energy with lard. They were randomized to fourgroups, as follows. The basal group continued this diet with nomodifications; a high calcium group received the basal diet supplementedwith CaCO₃ to increase dietary calcium by three-fold to 1.2%; a mediumdairy diet, in which 25% of the protein was replaced by non-fat dry milkand dietary calcium was increased to 1.2%; and a high dairy group inwhich 50% of the protein was replaced by non-fat dry milk, increasingcalcium to 2.4%. Food intake and spillage was measured daily, andanimals were weighed weekly. At the conclusion of the 6 week feedingperiod, animals were killed by exsanguination under isofluoraneanesthesia, and blood was collected via cardiac puncture for glucose andinsulin measurements. Fat pads (epididymal, perirenal, abdominal, andsubscapular) were dissected, immediately weighted, frozen in liquidnitrogen, and stored at −80° C. Fatty acid synthase activity and mRNAlevels were measured in abdominal fat.

[0056] Core Temperature

[0057] Core temperature was used as an indirect metabolic index todetermine whether any reduction in efficiency of conversion of goodenergy to body weight was accompanied by increased thermogenesis.Temperature was measured via a thermo-couple (Columbus Instruments,Columbus, Ohio). The probe was inserted a constant distance (1.8 cm)into the rectum of each animal. After stabilization (10 s), thetemperature was recorded every 5 s for 30 s. All temperaturemeasurements were made between 8:00 and 9:00 a.m.

[0058] Intracellular Calcium (Human Adipocytes)

[0059] Intracellular Ca²⁺ was determined fluorometrically as describedpreviously. Cells were washed with HEPES-buffered salt solution loadedwith fura-2-acetoxymethyl ester (10 μM) for 45 minutes at 37° C. in thedark with continuous shaking. Cells were then rinsed three times,resuspended, and intracellular Ca²⁺ was measured using dual excitation(340 and 380 nm)/single emission (510 nm) fluorometry. After theestablishment of a stable baseline, the response to 1,25-(OH)₂-D orparathyroid hormone (10 pM-100 nM) or their respective vehicles wasdetermined. Digitonin (25 μM) and Tris/EGTA (100 mM, pH 8.7) were usedto for calibration to calculate the final intracellular Ca²⁺.

[0060] Lipolysis

[0061] Adipocytes were incubated for 4 hours in the presence or absenceof forskolin (1 μM), and glycerol release into the culture medium wasmeasured to assess lipolysis. Glycerol release data was normalized forcellular protein.

[0062] Fatty Acid Synthase Activity and mRNA Levels

[0063] Immediately after death, adipose tissue was isolated and fattyacid synthase activity was measured in cytosolic extracts by measuringthe oxidation rate of NADPH. Enzyme activity was protein corrected usingCoomassie blue dye.

[0064] Total RNA was extracted by cesium chloride density gradient,electrophoresed, subjected to Northern blot analysis, and hybridizedwith a radiolabeled rat cDNA probe for fatty acid synthase usingstandard methods. Autoradiographs were quantitated densitometrically,and all blots were stripped and reprobed with β-actin as a loadingcontrol.

[0065] Statistical Analysis (In vitro and Animal Data)

[0066] All data are expressed as mean±SD. Data were evaluated forstatistical significance by one-way analysis of variance (ANOVA) or ttest, depending on the number of comparisons made. All data sets withmultiple comparisons were analyzed via ANOVA, followed by separation ofsignificantly different group means via test the least significantdifference using SPSS-PC (v. 8.0).

[0067] NHANES III Analysis

[0068] To determine whether the animal observations are relevant indefining a role for dietary calcium in modulating body composition atthe population level, an analysis of the National Health and NutritionExamination Survey (NHANES III) data set was conducted. This largecross-sectional survey conducted between 1988 and 1994 followed acomplex, four-stage probability sampling scheme designed to representthe entire U.S. civilian noninstitutionalized population over the age of2 months. Only adults completing all three phases of the study(interview, physical examination, and laboratory examination) wereincluded in this data analysis: respondents were excluded from thisanalysis if they could not provide complete, usable bodycomposition/anthropometric data (e.g., amputees and individuals wearingcasts), used insulin, or were pregnant, recently pregnant, or currentlybreast-feeding. Body composition was assessed using the anthropometricand bioelectrical impedance data collected during the physicalexamination, with percent body fat calculated using the regressionequations derived by Segal.

[0069] Odds ratios for percent body fat and corresponding 95% confidenceintervals were estimated by multiple logistic regression analysis with aRoust variance estimation method using SUDAAN (Shah, et al., SUDAANUser=s Manual, Release 7.0 (1996)). Point estimates for all parameterswere weighted to reflect the population distribution of each; varianceswere calculated using SUDAAN to take the complex sampling design intoaccount. Analyses were conducted separately for men and women, and allodds ratios were adjusted for age by including age in the model as acontinuous variable. Other covariates included in the model were caloricintake, race/ethnicity, and activity level. Characteristics of the studysample are shown in Tables 1 and 2. TABLE 1 Characteristics of NHANESIII Study sample^(a) Women Men (n = 380) (n = 7114) Age (years) 28.7 ±0.4 43.5 ± 0.44 Body mass index (kg/m²) 25.7 ± 0.4 26.6 ± 0.11 Body fat(%) 32.7 ± 0.6  25 ± 0.2 Calcium intake (mg/day) 720 ± 52 965 ± 15 Dairy product consumption 54.7 ± 3   51.4 ± 1   (monthly frequency)Energy intake (kcal/day) 1896 ± 68  2656 ± 28  Dietary fat (g/day) 74 ±4 102 ± 2 

[0070] TABLE 2 Quartiles of body fat for women and men in the NHANES IIIStudy^(a) % Body fat Quartile Women Men 1 22.82 ± 0.45 15.47 ± 0.14 230.54 ± 0.17 22.83 ± 0.06 3 34.85 ± 0.18 27.55 ± 0.06 4 42.49 ± 0.4434.08 ± 0.11

[0071] Results

[0072]FIG. 1 demonstrates that both 1,25-(OH)₂-D and parathyroid hormone(PTH) stimulate significant, sustained increases in intracellular Ca²⁺in primary cultures of human adipocytes (P<0.001: EC₅₀˜50 pM for1,25-(OH)₂-D and ˜10 nM for PTH). 1,25-(OH)₂-D treatment also resultedin marked (83%) inhibition of forskolin-stimulated lipolysis (P<0.001)in human adipocytes (FIG. 2). PTH treatment exerted little effect onlipolysis (data not shown) despite its stimulation of an intracellularCa²⁺ response, most likely as a result of an accompanying activation ofadenylate cyclase.

[0073] Treatment of aP2-transgenic-agouti mice with the high fat/highsucrose basal diet resulted in a weight gain of 24%, which was reducedby 26 and 29% by the high calcium and medium dairy diets, respectively(P<0.04), and further reduced by 39% by the high dairy diet (P<0.04;FIG. 3). These differences occurred despite the lack of any differencein food intake. Measurement of core temperature, an indirect metabolicindex, reflected these observations, with ˜0.5° C. increases in coretemperature in response to all three high calcium diets (P<0.03; FIG.4). This increase, coupled with the lack of difference in food intake,is indicative of a shift in efficiency of energy metabolism from energystorage to thermogenesis.

[0074] This shift in energy metabolism was evident in studies of fattyacid synthase, a key enzyme in de novo lipogenesis that is highlysensitive to regulation by nutrients and hormones. The basal diet causeda 2.6-fold increase in fatty acid synthase activity, and this effect wasmarkedly attenuated by all three high calcium diets (P<0.002; FIG. 5A).The diets caused corresponding decreases in adipocyte fatty acidsynthase mRNA, with a 27% reduction on the high calcium diet and a 51%reduction on the medium and high dairy diets (P <0.01; FIG. 5B).Adipocyte lipolysis responded to dietary manipulations in an inversefashion to the fatty acid synthase responses. The basal diet caused amarked (67%) suppression of lipolysis (P<0.0001); however, lipolysis wasstimulated 3 to 5.2-fold by the high calcium diets (P<0.01 5; FIG. 5C),with greater effects from the high dairy diets than from the highcalcium diet. Assessment of fat pad mass after 6 wk of dietary treatmentprovides further support for these findings. Table 3 demonstrates thatall three high calcium diets caused a 36% reduction in mass of theepididymal, abdominal, perirenal, and subscapular adipose tissuecompartments (P<0.001). Epididymal and subscapular fat pad mass wasreduced by ˜50% by all three diets, whereas the abdominal fat padsexhibited greater decreases on the medium and high dairy diets than onthe high calcium diet (P<0.001; Table 3). TABLE 3 Effects of calcium anddairy products on fat pad mass in transgenic mice expressing Agouti inadipose tissue under control of the aP2 promoter^(a) Basal High CalciumMedium dairy High dairy Abdominal (g) 2.239 ± 0.109 1.807 ± 0.082* 1.661 ± 0.127**  1.680 ± 0.113** Perirenal (g) 1.675 ± 0.124 1.271 ±0.098* 1.172 ± 0.128* 1.052 ± 0.094* Epididymal (g) 0.198 ± 0.036 0.110± 0.017* 0.110 ± 0.014* 0.097 ± 0.015* Subscapular (g) 1.592 ± 0.3180.680 ± 0.069* 0.663 ± 0.068* 0.639 ± 0.087* Sum^(b) (g) 5.703 ± 0.5483.649 ± 0.238* 3.705 ± 0.276* 3.787 ± 0.251*

[0075] Serial measurements of plasma glucose and insulin demonstrate adiabetogenic effect of the basal high fat/high sucrose/low calcium diet,with an increase in fasting glucose from 98±10 to 130±11 mg/dl (P<0.02)and a corresponding degree of compensatory hyperinsulinemia. Theseincreases were attenuated by the high calcium and medium dairy diets andprevented by the high dairy diet (FIG. 6).

[0076] Table 4 summarizes the NHANES III data analysis. Aftercontrolling for energy intake, activity level, age, race, and ethnicity,the odds ratio of being in the highest quartile of body fat was markedlyreduced from 1.00 for the first quartile of calcium intake to 0.75,0.40, and 0.16 for the second, third, and fourth quartiles, respectively(multiple R²=0.20; P=0.0009), in adult women. Similarly, the regressionmodel for males demonstrated an inverse relationship between calcium anddairy intakes and body fat (multiple R²=0.40;P=0.0006), although acomparable dose-responsive reduction in relative risk (odds ratio) byquartile of calcium intake was not evident from the model. TABLE 4Effects of dietary calcium, and dairy intake on the risk of being in thehighest quartile of body fat for women^(a) Quartile of Calcium DairyOdds ratio of calcium intake consumption being in the and dairy (mg/day:(servings, month: highest body consumption mean ± SEM) mean ± SEM) fatquartile 1 255 ± 20  14.4 ± 1.9 1.00 2 484 ± 13   38 ± 1.3 0.75 (0.13,4.22)^(b) 3 773 ± 28  57.2 ± 1.0 0.40 (0.01, 3.90)^(b) 4 1346 ± 113102.8 ± 3.6 0.16 (0.03, 0.88)^(b)

EXAMPLE 2 Effects of Dietary Calcium on Adipocyte Extracellular Calcium,Lipid Metabolism, and Weight and Fat Reduction Materials and Methods

[0077] Animals and Diets

[0078] aP2-agouti transgenic (aP2-a) mice were used as an animal modelin this study. The characterization of these animals has been describedpreviously (Mynatt, et al. (1997)). These transgenic mice express normalagouti protein specifically in adipose tissue under the control of theaP2 promoter (Mynatt, et al. (1997)), similar to the adipocyte-specifichuman pattern of agouti expression. These mice are useful as an animalmodel for the study of diet-induced obesity study, as they are not obeseon a standard AIN 93-G diet, but become obese in response tohyperinsulinemia induced by either exogenous insulin administration(Mynatt, et al. (1997)) or high sucrose diet (Zemel, et al. (1999)).

[0079] This study was divided into two 6-week stages. In the firststage, 60 6-week-old male aP2 mice were placed on a modified AIN 93-Gdiet with suboptimal calcium (0.4%), sucrose as the sole carbohydratesource and lard added to increase fat to 25% of energy. To evaluate thislow calcium/high fat/high sucrose diet-induced obesity in these animals,we monitored the body weight was monitored every 5 days and 8representative mice were euthanized for measurement of fat pad mass andadipocyte [Ca²⁺]_(i) at the end of first 6 week-basal diet feeding. Inthe second 6-week stage, the rest of mice that exhibited diet-inducedobesity were randomly assigned to five groups. One group was continuedad lib on the same low calcium (0.4%) diet with no modification, whilethe other four groups were maintained with energy restriction (70% of adlib) as follows. The mice in basal restriction group were placed on thebasal low calcium (0.4%) diet with Kcal-restriction. A high calciumenergy restricted group received the basal diet supplemented withcalcium increased to 1.2%. Two additional groups, termed medium dairyand high dairy, were fed modified basal diet in which either 25 or 50%of protein was replaced by non-fat dry milk, with total dietary calciumincreased to 1.2 or 2.4%, respectively. Diet was administrated daily andbody weight was monitored every five days. At the end of second stage,all mice were euthanized with beuthanasia (concentrated pentobarbitalwith phenytoin) and blood was obtained via cardiac puncture for insulinand glucose measurement. Fat pads (epididymal, perirenal, abdominal andsubscapular) were dissected, immediately weighed, frozen in liquidnitrogen, and stored at −80° C. Fatty acid synthase activity and mRNAlevels were measured in abdominal fat.

[0080] Core Temperature

[0081] Core temperature was used as an indirect metabolic index todetermine if dietary calcium regulates energy metabolism associated withincreased thermogenesis, an important contribution to energyexpenditure. Temperature was measured via a thermocouple (ColumbusInstruments, Columbus, Ohio) weekly (Kim, et al. (1996)). The probe wasinserted a constant distance (1.8 cm) into the rectum of each mouse.After stabilization (10 seconds), the temperature was recorded every 5seconds for 30 seconds. All core temperature measurements were performedbetween 8:00 and 9:00 A.M.

[0082] Mouse Adipocyte Intracellular Ca²⁺ ([Ca²⁺]_(i)) Measurement

[0083] Isolated mouse adipocytes were prepared from mouse abdominal fatdepots as previously described (Shi, et al. (1999)) with slightmodification. Briefly, adipose tissue was first washed several timeswith Hank's Balanced Salt Solution, minced into small pieces anddigested with 0.8 mg/ml type I collagenase in a shaking water bath at37° C. for 30 min. Adipocytes were then filtered through a sterile 500μm nylon mesh and cultured in Dulbecco's Modified Eagle's Medium (DMEM)supplemented with 1% fetal bovine serum (FBS). Cells were cultured insuspension and maintained in a thin layer at the top of culture mediafor two hours for cell recovery.

[0084] [Ca²⁺]_(i) in isolated mouse adipocytes was measured using afura-2 dual wavelength fluorescence imaging system. Prior to [Ca²⁺]_(i)measurement, adipocytes were pre-incubated in serum-free medium for 2hrs and rinsed with Hepes Balanced Salt Solution (HBSS) containing thefollowing components (in mmol/L): NaCl 138, CaCl₂ 1.8, MgSO₄ 0.8,NaH₂PO₄ 0.9, NaHCO₃ 4, glucose 5, glutamine 6, Hepes 20, and bovineserum albumin 1%. Adipocytes were loaded with fura-2 acetoxymethyl ester(AM) (10 μmol/L) in the same buffer for 1 hr at 37° C. in a darkincubator with 5% CO₂. To remove extracellular dye, adipocytes wererinsed with HBSS 3 times and then post-incubated at room temperature foran additional 30 min for complete hydrolysis of cytoplasmic fura-2 AM. Athin layer of adipocytes was plated in 35 mm dished with glasscoverslips (P35G-0-14-C, MatTek Corporation). The dishes with dye-loadedcells were mounted on the stage of Nikon TMS-F fluorescence invertedmicroscope with a Cohu 4915 CCD camera. Fluorescent images were capturedalternatively at excitation wavelength of 340 and 380 nM with anemission wavelength of 520 nM. [Ca²⁺]_(i) was calculated using a ratioequation as described previously(Grynkiewicz, et al. (1985)).

[0085] Fatty Acid Synthase (FAS) Activity Assay

[0086] FAS activity was determined spectrophotometrically in crudecytosolic extracts of mouse adipose tissue as previously described(Jones, et al. (1997)). Mouse abdominal fat pads were homogenized in 250mmol/L sucrose solution containing 1 mmol/Lethylenediamine-tetraacticacid (EDTA), 1 mmol/L dithiothreitol (DTT),and 100 μmol/L phenylmethylsulfonyl fluoride (PMSF) (pH 7.4). Homogenatewas centrifuged at 18,500×g for 1 hr and the infranatant was used formeasuring oxidation rate of NADPH.

[0087] Lipolysis Assay

[0088] Following the sacrifice, mouse perirenal adipose tissue wasimmediately dissected and incubated for four hours. Glycerol releasedinto the culture medium was determined as an indicator of lipolysis,using a one-step enzymatic fluorometric method as previously described(Boobis, et al. (1983)).

[0089] Northern Blot Analysis

[0090] Northern blot analysis was conducted as described (Shi, et al.(1999)). Total RNA from mouse abdominal adipose tissue was extractedusing CsCl₂ density centrifugation, run in 1% agarose gel andtransferred to nylon membrane (New England Nuclear, Boston, Mass.). Themembrane was hybridized with uncoupling protein 2 (UCP2) or FAS cDNAprobes that were radiolabeled using a random primer method. Unboundprobe was removed by rinsing the membrane with 2×SSC/0.1% SDS for 30 minat room temperature and 0.1×SSC/0.1% SDS for 45 min at 55° C. Finally,the membrane was exposed to X-ray film (New England Nuclear, Boston,Mass.) at −80° C. All membranes were stripped and reprobed with β-actinas loading control.

[0091] Statistical Analysis

[0092] All data are expressed as mean±SE and evaluated for statisticalsignificance by one way Analysis of Variance (ANOVA) using SPSS (SPSSInc, Chicago, Ill.). A p value<0.05 is considered significant.

[0093] Results

[0094] To establish the animal model of diet-induced obesity inaP2-agouti transgenic (aP2-a) mice, mice were placed on low calcium(0.4%)/high fat/high sucrose diet for 6 weeks. Administration of aP2-amice with this diet resulted in a 2-fold increase in adipocyte[Ca²⁺]_(i) (128 ±18 vs. 267±15 nM, p<0.001, lower panel, FIG. 7), with acorresponding body weight gain of 27% (p<0.001, upper panel, FIG. 7) andtwo fold increase in total fat pad mass (p<0.001, middle panel, FIG. 7),demonstrating that diet-induced dysregulation of adipocyte [Ca²⁺]_(i) isassociated with increased adiposity in aP2-a mice.

[0095] The role of dietary calcium in facilitating weight loss incalorically restricted obese aP2-a mice was evaluated. These obese aP2-amice were maintained either on the same low calcium basal diet ad lib oron the Kcal-restricted (70% of ad lib) diets supplemented with orwithout dairy or CaCO₃ as dietary calcium supplementary sources. FIG. 8illustrates that all three calcium diets, including high calcium diet(1.2% Ca²⁺ derived from CaCO₃), medium dairy diet (1.2% Ca²⁺ derivedfrom non-fat dry milk replacing 25% of protein) and high dairy diet(2.4% Ca²⁺ derived from non-fat dry milk replacing 50% of protein),caused a 50% decrease in adipocyte [Ca²⁺]_(i) (p<0.001, FIG. 8), while[Ca²⁺]_(i) in adipocytes from mice maintained on the Kcal-restrictedbasal low calcium diet remained at the same elevated level as that of adlib animals.

[0096] An examination was conducted as to whether a dietarycalcium-induced decrease in adipocyte [Ca²⁺]_(i) facilitated the bodyweight loss and fat pad mass reduction. As shown in FIG. 8, energyrestriction resulted in a body weight loss by 11% (p<0.001, FIG. 8),compared to ad lib group. However, markedly greater weight reductions of19, 25, 29% were observed in the high calcium, medium and high dairygroups, respectively (p<0.01 vs. basal energy-restricted group, FIG. 8).Consistent with this, energy restriction caused an only 8% decrease infat pad mass, compared to basal diet ad lib group, while the highcalcium diet caused a 42% decrease (p <0.001, FIG. 8), which was furtherreduced by 60 and 69% by the medium and high dairy diets (p<0.001 vs.basal energy-restricted group, FIG. 8), respectively.

[0097]FIG. 9 demonstrates that the high calcium diet caused a 35%decrease in fatty acid synthase (FAS) activity (p<0.05 vs. basalenergy-restricted group), which was further reduced by 63 and 62% by themedium and high dairy diets (p<0.05), respectively. Similarly, the threehigh calcium diets caused corresponding decreases in adipocyte FAS mRNA,with 61%, 72% and 81% reductions on high calcium, medium dairy and highdairy diets, respectively (p<0.05 vs. basal energy-restricted group,FIG. 9). Increasing dietary calcium caused a corresponding increase inlipolysis. Although the basal energy restricted diet did not affectadipocyte lipolysis, the high calcium diet caused 77% stimulation inlipolysis (p<0.05. FIG. 9), which was further increased by 2 fold in themedium and high dairy diet groups (p<0.05 vs. basal energy-restrictedgroup, FIG. 9). Increased lipolysis, coupled with decreased lipogenesis,may represent a metabolic state in which the efficiency of energymetabolism is shifted from energy storage to energy expenditure.

[0098] This shift in energy metabolism was further confirmed by dietarycalcium-induced increase in core temperature. All three high calciumdiets exerted stimulatory effects on core temperature, with 0.48° C.,0.57° C. and 0.67° C. increases on the high calcium, medium dairy andhigh dairy diets, respectively (p<0.05), while the basal energyrestricted diet did not affect on core temperature. A possiblephysiological basis underlying the increased core temperature is thatthe expression of uncoupling protein 2 (UCP2), which has been implicatedin thermogenesis (Fleury, et al. (1997); Gimeno, et al. (1997)), wasup-regulated in white adipose tissue, with 80% increase on all threehigh calcium diets (p<0.05, FIG. 10). This demonstrates that dietarycalcium may also modulate energy metabolism associated withthermogenesis. Accordingly, a coordinated up-regulation of lipolysis anddown-regulation of lipogenesis, coupled with increased thermogenesis,may serve to shift the energy metabolism from energy storage to energyexpenditure on high calcium diets.

[0099] The basal low calcium diet exerted a hyperinsulinemic effect,with a 2-fold increase in plasma insulin levels (p<0.001, FIG. 11). Themice maintained on this basal diet ad lib exhibited sustained levels ofhyperinsulinemia, while energy restriction per se reduced plasma insulinby approximately 50%. The high calcium diets did not furthersignificantly affect insulin levels, although there was anon-significant trend towards further reductions (FIG. 11).

EXAMPLE 3 Methods of Administering a High Calcium Diet

[0100] The subject invention provides methods useful in diagnosing,treating, and/or monitoring obesity. One method of the subject inventioncomprises the following steps:

[0101] a. determining the weight and, optionally, the height of anindividual;

[0102] b. comparing the weight, or optionally the weight/height ratio,of the individual to established norms;

[0103] c. optionally, classifying the obesity of the individual;

[0104] d. optionally providing the individual with information relatingto the benefits of maintaining a normal weight, or a normalweight/height ratio; and

[0105] e. providing the individual with a dietary plan containing highlevels of calcium and, optionally printed matter disclosing theobesity-control benefits of a high calcium diet.

[0106] The subject invention also provides computer implemented methodsof diagnosing, treating, and/or monitoring obesity. The methods of thisembodiment of the subject invention can comprise the following steps:

[0107] a. determining the weight and, optionally, the height of anindividual and inputting these values into a computer system;

[0108] b. optionally calculating the weight/height ratio of theindividual;

[0109] c. comparing the weight, or optionally the weight/height ratio,of the individual to established norms contained in a weight and/orweight/height database available to the computer;

[0110] d. optionally classifying the obesity of the individual;

[0111] e. optionally providing the individual with information relatingto the benefits maintaining a normal weight, or optionally a normalweight/height ratio; and

[0112] f. providing the individual a dietary plan containing high levelsof calcium and, optionally printed matter disclosing the obesity-controlbenefits of a high calcium diet.

[0113] g. optionally monitoring the progress of the individual.

[0114] The subject invention also provides computer implemented methodsof diagnosing, treating, and/or monitoring obesity over a communicationnetwork. The methods of this embodiment of the subject invention cancomprise the following steps:

[0115] a. obtaining weight and, optionally, height data from anindividual by input of the data on a web page;

[0116] b. optionally calculating the weight/height ratio of theindividual in a computer connected to the communication network;

[0117] c. comparing the weight, or optionally the weight/height ratio,of the individual to established norms contained in a weight and/orweight/height database available to said computer;

[0118] d. optionally classifying the obesity of the individual;

[0119] e. optionally providing the individual with information relatingto the benefits maintaining a normal weight, or optionally a normalweight/height ratio; and

[0120] f. providing the individual a dietary plan containing high levelsof calcium and, optionally, information regarding the obesity-controlbenefits of a high calcium diet.

[0121] This method may further provide requesting verification that theweight and height values inputted by the individual are correct. Themethod may also provide the classification of obesity, informationrelating to the benefits maintaining a normal weight, or optionally anormal weight/height ratio, providing the individual a dietary plancontaining high levels of calcium and/or information regarding benefitsof a high calcium diet to the individual only at the request of theindividual. The communication network may be the Internet, an intranet,LAN, WAN, a real private network, or two or more computers connectedelectronically.

[0122] It should be understood that the examples and embodimentsdescribed herein are for illustrative purposes only and that variousmodifications or changes in light thereof will be suggested to personsskilled in the art and are to be included within the spirit and purviewof this application and the appended claims.

We claim:
 1. An article of manufacture comprising a calcium-containingdietary product and a description of a health effect of consuming acalcium-containing dietary product, the described health effect beingrelated to calcium and selected from the group consisting of one or moreof metabolic consumption of adipose tissue, metabolic change, regulationof body weight, weight loss, reduced weight gain, fat loss, and reducedfat gain.
 2. The article of claim 1, wherein the description is in theform of printed material.
 3. The article of manufacture of claim 1,wherein the product is packaged and the description is part of thepackage.
 4. The article of manufacture of claim 1, wherein thedescription directly accompanies the product.
 5. The article ofmanufacture of claim 1, wherein the description is imprinted on theproduct.
 6. The article of manufacture of claim 1, wherein thedescription indicates the amounts of calcium contained within theproduct, and recommended levels of calcium intake for regulation of bodyweight.
 7. The article of manufacture of claim 1, wherein the describedeffects comprise inducing a metabolic change in an individual.
 8. Thearticle of manufacture of claim 7, wherein the metabolic changecomprises decreasing intracellular calcium concentrations ([Ca²⁺]_(i)),stimulating lipolysis, inhibiting lipogenesis, increasing the expressionof white adipose tissue uncoupling protein 2 (UCP2), reducing seruminsulin levels, thermogenesis, and/or decreasing the levels ofcalcitrophic hormones.
 9. The article of manufacture of claim 1, whereinthe described effects comprise reducing or attenuating obesity.
 10. Thearticle of manufacture of claim 1, wherein the described effectscomprise attenuating weight gain and/or adiposity in children.
 11. Thearticle of claim 1, wherein the product comprises a dairy product. 12.The article of claim 1, wherein the product is selected from the groupconsisting of milk, yogurt, and cheeses.
 13. The article of claim 1,wherein the product comprises a non-dairy foodstuff naturally high incalcium.
 14. The article of claim 1, wherein the product comprises afood fortified with calcium.
 15. The article of claim 1, wherein theproduct is a dietary supplement.
 16. The article of claim 1, wherein theproduct is in the form of a liquid.
 17. The article of manufacture ofclaim 1, wherein the product is cereal and the printed material isprinted on the cereal box.
 18. The article of manufacture of claim 1,wherein the product is milk and the printed material is printed on themilk container.
 19. The article of manufacture of claim 1, wherein theproduct is cheese and the printed material is printed on the cheesepackage.
 20. The article of manufacture of claim 1, wherein the productis yogurt and the printed material is printed on the yogurt container.21. The article of claim 1, wherein the product is food for a pet. 22.The article of claim 1 wherein the therapeutically effective amountdescribed is at least about 1000 mg/day of calcium.
 23. The article ofclaim 1 wherein the therapeutically effective amount described is atleast about 1346 mg/day of calcium.
 24. An article of manufacture usefulin stimulating the metabolic consumption of adipose tissue, comprising adairy product and printed material describing the obesity regulatingeffects of consuming more than suboptimal amounts of dairy products. 25.The article of claim 24 wherein the more than suboptimal amountdescribed is at least about 57 portions of dairy per month.
 26. Anarticle of manufacture useful in stimulating the metabolic consumptionof adipose tissue, comprising calcium-containing products and printedmaterials disclosing the weight loss advantages of high calcium diets.27. The article of manufacture of claim 26, wherein the printedmaterials are in the form of pamphlets.
 28. The article of manufactureof claim 26, wherein printed material is embossed or imprinted on thefoodstuff and indicates the amounts of calcium contained within thefoodstuff, recommended levels of calcium intake necessary for themetabolically assisted loss of adipose tissue, recommended BMI values,or recommended heights and weights for individuals.